Genetic knockouts and knockins in human somatic cells

Rago, Carlo; Vogelstein, Bert; Bunz, Fred

doi:10.1038/nprot.2007.408

Cited by 102 publications

(121 citation statements)

References 59 publications

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“…1A). This strategy was applied to the colorectal cancer cell line DLD-1, which has been extensively used for cell cycle analysis and has been shown to exhibit a high rate of recombinant adeno-associated virus (rAAV)-mediated gene targeting (19). DLD-1 cells are near diploid and harbor two wild-type CHK1 alleles.…”

Section: Resultsmentioning

confidence: 99%

“…Homology arms were ligated to the SEPT selectable marker (30) to create the AAV shuttle constructs pAAV-Chk1S317A and pAAVChk1S345A. Virus production, infection, selection, the screening of homologous integrants, and Cre-mediated excision of the selectable marker was performed as described (19). A second round of gene targeting was performed with an rAAV-based knockout construct designed to disrupt exon 3, pAAV-⌬Chk1.…”

Section: Methodsmentioning

confidence: 99%

“…A second round of gene targeting was performed with an rAAV-based knockout construct designed to disrupt exon 3, pAAV-⌬Chk1. RNA was extracted from identified knockout clones (19) with the RNAeasy kit (Invitrogen), and cDNA amplification was performed with SuperScript II (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

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Essential function of Chk1 can be uncoupled from DNA damage checkpoint and replication control

Wilsker

Petermann

Helleday

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Chk1 is widely known as a DNA damage checkpoint signaling protein. Unlike many other checkpoint proteins, Chk1 also plays an essential but poorly defined role in the proliferation of unperturbed cells. Activation of Chk1 after DNA damage is known to require the phosphorylation of several C-terminal residues, including the highly conserved S317 and S345 sites. To evaluate the respective roles of these individual sites and assess their contribution to the functions of Chk1, we used a gene targeting approach to introduce point mutations into the endogenous human CHK1 locus. We report that the essential and nonessential functions of Chk1 are regulated through distinct phosphorylation events and can be genetically uncoupled. The DNA damage response function of Chk1 was nonessential. Targeted mutation of S317 abrogated G 2/M checkpoint activation, prevented subsequent phosphorylation of Chk1, impaired efficient progression of DNA replication forks, and increased fork stalling, but did not impact viability. Thus, the nonessential DNA damage response function of Chk1 could be unambiguously linked to its role in DNA replication control. In contrast, a CHK1 allele with mutated S345 did not support viability, indicating an essential role for this residue during the unperturbed cell cycle. A distinct, physiologic mode of S345 phosphorylation, initiated at the centrosome during unperturbed mitosis was independent of codon 317 status and mechanistically distinct from the ordered and sequential phosphorylation of serine residues on Chk1 induced by DNA damage. Our findings suggest an essential regulatory role for Chk1 phosphorylation during mitotic progression.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Essential function of Chk1 can be uncoupled from DNA damage checkpoint and replication control

Wilsker

Petermann

Helleday

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

120

131

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“…Disruption of the CLS1 locus in HCT116 cells was based on previously published procedures 47,48 with some modifications. Vectors were a gift of R. Youle.…”

Section: Methodsmentioning

confidence: 99%

Cardiolipin or MTCH2 can serve as tBID receptors during apoptosis

et al. 2016

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During apoptosis, proapoptotic BAX and BAK trigger mitochondrial outer membrane (MOM) permeabilization by a mechanism that is not yet fully understood. BH3-only proteins such as tBID, together with lipids of the MOM, are thought to play a key role in BAX and BAK activation. In particular, cardiolipin (CL) has been shown to stimulate tBID-induced BAX activation in vitro. However, it is still unclear whether this process also relies on CL in the cell, or whether it is more dependent on MTCH2, a proposed receptor for tBID present in the MOM. To address this issue, we deleted both alleles of cardiolipin synthase in human HCT116 cells by homologous recombination, which resulted in a complete absence of CL. The CL-deficient cells were fully viable in glucose but displayed impaired oxidative phosphorylation and an inability to grow in galactose. Using these cells, we found that CL was not required for either tBID-induced BAX activation, or for apoptosis in response to treatment with TRAIL. Downregulation of MTCH2 in HCT116 cells also failed to prevent recruitment of tBID to mitochondria in apoptotic conditions. However, when both CL and MTCH2 were depleted, a significant reduction in tBID recruitment was observed, suggesting that in HCT116 cells, CL and MTCH2 can have redundant functions in this process.

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“…These results suggest that the C-terminal Ring finger domain is required for the ability of MIB1 to npg activate NF-κB, while the N-terminal Zinc finger domain of MIB1 contributes to its full activity. To investigate the mechanisms by which MIB1 activates NF-κB, we attempted to generate MIB1 knockout cells, utilizing a recently reported method [13]. Following these procedures, we generated three independent MIB1 gene knockout clones in human colon cancer HCT116 cells.…”

Section: Dear Editormentioning

confidence: 99%